During the initial period of this grant we have: (1) purchased supplies; (2) tested endotoxin contamination in all media and reagents; (3) set up in vitro cytotoxicity assay and investigated the time and dose dependency of lymphokine/lipopolysaccharide (LPS) activation; and (4) attempted to establish the optimal experimental conditions for the determination of intracellular calcium with aequorin. One of the major problems we have faced is related to the fact that RAW-264 macrophage-like cell lines used in our study have shown a very low rate of triggering of cytolytic responses upon sequential lymphokine/LPS activation. Since methods of determination of intracellular calcium, which we are planning to use, are not feasible for monitoring of slow changes in calcium (this kind of change may be expected in RAW-264 cells judging by the slow rate of their cytolytic responses), in the future we will probably have to use normal macrophages instead of RAW-264 cells. The second problem is related to the determination of intracellular calcium with luminescence probe aequorin. The hypo-osmotic shock treatment (HOST) used for introducing aequorin into cells completely abolishes the responsiveness of RAW-264 cells to lymphokine/LPS activation. The responsiveness is regained after several hours, but by then the content of aequorin in cells is negligible. As a solution to this problem, we intend to: (1) investigate the effect of HOST procedure on cytolytic responses and the level of aequorin incorporation in normal peritoneal or bone marrow macrophages; (2) apply the modified procedure for aequorin incorporation based on the combination of hyperosmotic/hypoosmotic treatment of cells; and (3) monitor changes in intracellular calcium using the novel method, which utilizes the fluorescence probe quin-2. (MB)